Since the introduction of insulin in the 1920's, continuous strides have been made to improve the treatment off diabetes mellitus. Major advances have been made in insulin purity and availability. Various formulations with different time-actions have also been developed. Despite these improvements, subcutaneous injection therapy still falls short of providing the patient with convenient regulation and normalized glycemic control. Frequent excursions from normal glycemia levels over a patient's lifetime lead to hyper- or hypoglycemia, and long term complications including retinopathy, neuropathy, nephropathy, and micro- and macroangiopathy.
To help avoid extreme glycemic levels, diabetics often practice multiple injection therapy whereby insulin is administered with each meal. However, this therapy has not yet been optimized. The most rapid-acting insulin commercially available peaks too late after injection and lasts too long to optimally control glucose levels. Recently, considerable effort has been devoted to create insulin formulations and insulin analog formulations that alter the kinetics of the subcutaneous absorption process.
Because all commercial pharmaceutical formulations of insulin contain insulin in the self-associated state and predominately in the zinc-hexamer form, it is believed that the rate-limiting step for the absorption of insulin from the subcutaneous injection depot to the bloodstream is the dissociation of the self-aggregated insulin hexamer. To accelerate this absorption process, monomeric insulin analogs have been developed. These monomeric analogs possess a comparatively more rapid onset of activity than insulin while retaining the biological activity of native human insulin. They provide a rapid absorption to place injection time and peak action of insulin into closer proximity with postprandial glucose excursion associated in the response to a meal.
The present invention provides a novel process of preparing crystals of one such monomeric analog, Lys.sup.B28 Pro.sup.B29 -human insulin (Lys.sup.B28 Pro.sup.B29 -hI). Lys.sup.B28 Pro.sup.B29 -hI is disclosed in U.S. patent application Ser. No. 07/388,201 (EPO publication number 383 472). However, U.S. patent application Ser. No. 07/388,201 does not disclose a commercially viable process of preparing crystalline Lys.sup.B28 Pro.sup.B29 -hI.
The crystallization of insulin is well known in the art. Initial discoveries date back to 1926 when Abel crystallized insulin in the isoelectric region from a solution buffered with brucine, pyridine, and ammonium acetate. Abel J. J., Proc. Nat'l Acad. Sci. U.S. 12: 132 (1926). Peterson, et al. in U.S. Pat. No. 2,920,104 describe insulin crystals and preparations and processes for producing them. R. L. Jackson, U.S. Pat. No. 3,719,655, discloses a process for preparing ammonium and alkali metal salts of insulin. The process comprises adjusting the basicity of a pork or beef insulin containing solution comprising pork or beef insulin and a cation selected from the group consisting of an alkali metal cation and the ammonium cation at a concentration of 0.2 molar to 1.0 molar to about pH 7.2 to about pH 10.0 with an alkali metal or ammonium base. The process has evolved commercially as the "8.2 process," that is the maximum yield of the insulin crystals is at pH 8.2. Most significantly, when Lys.sup.B28 Pro.sup.B29 -human insulin is subjected to the pH 8.2 conditions described in Jackson to form sodium crystals, no such crystallization occurs.
The present invention provides specific conditions under which monomeric Lys.sup.B28 Pro.sup.B29 -human insulin will associate and crystallize. Accordingly, the invention provides a process of crystallizing Lys.sup.B28 Pro.sup.B29 -hI. The process prepares high quality, high yield alkali metal or ammonium salts as crystals on a large scale. The crystals provide a stable, solid form of the molecule. Crystalline solids are particularly advantageous because they are more easily characterized, purified, and more pharmaceutically elegant than solids that are amorphous. The process is suitable for commercial application.